Serine Palmitoyl Transferase (SPT) is the enzyme that carries out the condensation reaction between Serine and Palmitoyl Co-A that is known as the committed step of sphingolipid biosynthesis. Dysregulation of this important pathway through its downstream sphingolipid metabolites has been implicated in a variety of human diseases, including diabetes, metabolic syndrome, neuropathy, and cancer, as well as several key regulatory pathways such as apoptosis, inflammatory responses, and autophagy. Recent discoveries from several laboratories, including the mentor's, have demonstrated SPT exhibits substrate promiscuity, both with other non-canonical amino acids as well as fatty acids, which has led to the uncovering of novel sphingolipid metabolite by-products of this promiscuity such as deoxysphinganine and deoxydemethylsphinganine. The long-term goal of this project is to establish signaling and biological functions for novel sphingolipids in Saccharomyces cerevisiae. We have now developed novel mass spectral-based methods to allow for high sensitivity and accuracy detection and quantitation of this emerging new class of sphingolipids. Preliminary data suggests there may be a regulatory relationship between sphingolipids and amino acid availability that extends beyond the canonical Serine and Palmitoyl Co-A to other amino acids such as Alanine and Glycine, and other Fatty Acids including Stearate. This proposal's main focus will be centered on the characterization of the novel sphingolipid metabolites and exploration of SPT's ability to coordinate amino acid metabolism, and will address the following aims: 1) Development of MS detection methodology for detection and quantitation of novel SLs. We will use Multiple Reaction Monitoring on a triple quad mass spectrometer in conjunction with high quality authentic SL standards and SILAC-like metabolic labeling of novel SLs to identify and measure relative levels of novel SL metabolites. 2) To characterize the metabolism and regulation of these novel SLs by non-Serine amino acids. We will examine production of novel metabolites in response to amino acid dosage, and carry out time-course studies to determine rate of synthesis of these metabolites. High amino acid dose, concomitant with treatment with inhibitors of SL synthesis inhibitor, will be used to track their potential metabolism into SLs and ceramides. 3) To define roles for novel SLs in amino acid metabolism regulation. We will utilize microarray approaches to determine genes involved in amino acid metabolism that are regulated by these novel SL metabolites. Using amino acid addition, heat, or myriocin treatment, we will validate which subset of these genes are also involved in SL metabolism. The amalgamation of these results will serve to define novel SLs and their potential roles in signaling, and to define their biological functions in yeast. These methodologies, including MS quantitative techniques, as well as metabolic characterization, can later be extrapolated and applied to exploring parallels in mammalian systems.